Method for synthesizing single-stranded stem-loop DNA&#39;s the products and uses therefore

ABSTRACT

A method of synthesis of new and useful single-stranded DNAs which have a stem-loop configuration (ss-slDNA). The method is an in vivo or an in vitro synthesis. Replicating vehicles which produce these ss-slDNAs. The ss-slDNAs are described. Uses for these slDNAs are disclosed. They can be used for introducing random mutations, they lend themselves for replication by a variant of the PCR method. They can also be used for regulating gene function. Other uses are disclosed.

This application is a continuation of application Ser. No. 08/024,676filed Mar. 1, 1993 now abandoned, which is a CIP of Ser. No. 07/753,111Aug. 30, 1991, now abandoned.

FIELD OF THE INVENTION

The invention concerns the field of recombinant DNA. More particularly,the invention relates to a method of synthesis of new and usefulsingle-stranded DNA which has a stem-loop configuration (ss-slDNA). Theinvention relates to an in vitro and in vivo method of synthesis.Further, the invention relates to the replicating vehicle which producesthese ss-slDNAs. Moreover, the invention relates these novel structuresand discloses uses for these structures. There is described a method foramplifying ss-slDNAs with or without genes encoding a target protein.Moreover, the invention discloses a method for regulating gene functionby the use of ss-slDNA.

BACKGROUND

Duplication of part of the genome is known to occur via an RNAintermediate which is reverse-transcribed into complementary DNA (cDNA)by reverse transcriptase. For a review, see Weiner et al., Ann. Rev.Biochem., 55, 631 (1986). The consequential reverse flow of geneticinformation is considered to have played a major role in theevolutionary diversification of eukaryotic genomes. A similar mechanismmay very well have been responsible for genomic evolution in procaryotesin the light of the recent discoveries of bacterial transcriptases. SeeInouye and Inouye, TIBS, 16, 18 (1991a) and Inouye and Inouye, Ann. Rev.Microbiol., 45, 163 (1991b). Gene duplication through cDNA synthesis byreverse transcriptase is believed to have played an important role indiversification of genomes during evolution.

The invention arose in connection with basic research related to genomeevolution. The synthesis of a unique ss-slDNA during plasmid DNAreplication was demonstrated. It may be speculated that slDNA productionmay be widely prevalent during both procaryotic as well as eucaryoticchromosomal DNA replication. The chromosomal genetic elements followedby IR structures may always be subject to duplication into slDNA at afrequency depending on the stability of the IR structure and theproperty of the polymerase(s).

It has been shown that there are many inverted repeat (IR) structures(approximately 1,000 copies in E. coli), known as REPs for repetitiveextragenic palindromic sequences or PUs for palindromic units. Higginset al., Nature, 298, 760 (1982), Gilson et al., EMBO. J., 3, 1417 (1984)and Gilson et al., Nucl. Acids Res., 19, 1375 (1991). These structuresappear to be associated with specific cellular components including DNApolymerase 1, and may be playing a significant role in chromosomalorganization. Gilson et al., Nucl. Acids Res. 18, 3941 (1990) and Gilsonet al., EMBO. J., 3, 1417 (1984). It should also be noted thatapproximately 6% of the human genome are occupied with elements calledAlu whose transcriptional products have been shown to containsubstantial secondary structures. Simmett et al., J. Biol. Chem., 266,8675 (1991).

Since slDNA synthesis does not require RNA intermediates nor reversetranscriptase activity in contrast to that of cDNA synthesis, slDNA maybe more frequently produced than cDNA. Thus, slDNAs might have played amajor role similar to cDNA in the genomic evolution of both procaryotesand eucaryotes by duplicating genetic elements which then were dispersedor rearranged within the genome.

SUMMARY OF THE INVENTION

In accordance with the present invention, a fundamental finding has beenmade. It has been found that portions of the genome can be directlyduplicated from the genome. This gene duplication it has been found,requires neither an RNA intermediate nor reverse transcriptase andoccurs during DNA replication.

Briefly described, the invention provides a method (or process) forsynthesizing a novel and useful single-stranded DNA (ssDNA) molecule.The method involves using a DNA inverted repeat (IR) and necessarycomponents to start and synthesize a single-stranded structure. Theinvention also provides a system for synthesizing such ssDNA from andwith the necessary components including a DNA inverted repeat. Theinvention further provides competent replicating vehicles which includeall the necessary elements to synthesize the novel ssDNA molecules. Theinvention also provides novel and useful ssDNA molecules which form aunique structure. The structure has a stem constituted of duplexed DNAof complementary bases; the stem terminates at one of its ends by twotermini, respectively a 3' and 5' terminus, and at the other end by thessDNA forming a loop.

The invention contemplates carrying out the method and the systems invitro or in vivo.

Various uses of the new molecules are described, including a method forproviding random mutations in genes with the aim of generating proteinswith improved or novel biological properties. Another interestingcontemplated use is to integrate the ssDNA molecules of the inventioninto DNA to generate a triplex DNA of increased stability. Another useis to use the ssDNA of this invention as antisense DNA. Another use isthe application of the polymerase chain reaction (PCR) using a singleprimer.

The invention and its several embodiments are described in greaterdetail hereinafter.

By the method of the invention there is produced a ssDNA molecule whosestructure comprises a stem portion of duplexed DNA of annealedcomplementary bases within the ssDNA, which stem forms, at one of itsends, the 5' and 3' termini of the ssDNA molecule and, at the other end,a loop, which is constituted of a single-strand of non-annealed baseswhich joins the two single-strands of the stem. In a specificembodiment, strand ending in the 3' terminus is longer than the otherstrand. The slDNAs may include DNA segments capable of encoding aprotein, more particularly any gene(s). The gene will be located betweenthe loop and the termini. The gene may be a gene carrying a mutation(s).

A mechanism postulated for the synthesis of slDNAs is illustrated inFIG. 6A and B. The synthesis is believed to involve, in summary, thefollowing course: DNA is initiated at the origin of replication (OR),replication of a first strand (or "the" or "a" strand) then proceedsusing one of the strands of the double-stranded DNA as template (by thesame mechanism as chromosomal DNA replication (see Tomizawa et al.,Proc. Natl. Acad. Sci. USA, 74, 1865 (1977)), proceeding through the IRstructure resulting in the replication of the entire plasmid genome whena plasmid is used. However (as is described in greater detailhereinafter), part of the first strand forms a loop structure as thefirst strand synthesis is disrupted or terminated within the IR (FIG. 6,from steps 2 to 3). The loop forms a short duplexed region whichfunctions as priming site for continuing DNA synthesis. DNA chainelongation resumes from the newly formed 3' end, now forming the secondstrand (or "the other" strand) utilizing the nascent first strand as atemplate. An alternative (or concurrent) postalated synthesis course isdescribed hereinafter. Thus, the direction of DNA synthesis is reversedby template switching to duplicate the DNA fragment (FIG. 6, from steps4 to 5). The newly synthesized slDNA molecule dissociates itself fromthe parent template strands which undergo another round of replicationto form another slDNA. The slDNA is isolated, if necessary, purified.

In another embodiment of the invention, there is provided a DNAself-replicating vehicle, e.g., a plasmid into which there has beeninserted a DNA fragment which contains an inverted repeat (IR)structure, the DNA fragment which will serve as template for synthesisof the ssDNA and a suitable priming site, e.g., an origin of replication(OR), such as the E. coli origin of replication (ColE1). The IR issituated downstream of the OR. The self-replicating vehicle isreplicated in a suitable host, e.g., an E. coli. The host will containat least one DNA polymerase to synthesize the ssDNA from the template.In this specific embodiment, it is presumed that there are twopolymerases, a first DNA polymerase contributing to the elongation of aportion of the ssDNA from the OR to the IR, the first strand, and asecond DNA polymerase which synthesizes the balance or second strand ofthe ssDNA strand. As the synthesis of the first strand terminates,complementary bases anneal to form a single-stranded non-annealed loop.The synthesis of the second strand then takes place. A new DNA structureis synthesized which is constituted of a duplexed DNA stem and asingle-stranded loop structure at the opposite end. For this new DNAstructure the term "stem-loop" or "slDNA" has been coined.

Another specific embodiment provides a replicating vehicle in which apromoter, specifically the lac promoter-operator has been deleted. AnslDNA was nonetheless produced, supporting the synthesis model proposed,as described further herein.

The plasmid may be constructed to contain any selected DNA sequencecapable of encoding a protein between the priming site and the IR inwhich event the slDNA synthesized will contain the DNA sequence or amutation thereof.

This invention provides a method for regulating gene function by the useof slDNA.

The slDNAs of the invention need not be synthesized by aself-replication vehicle, but may be synthesized in an appropriate invitro system constituted of a any segment of DNA, linear or not, whichcontains any IR and the necessary elements to synthesize the ssDNA. Suchsystem is described hereinafter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a plasmid of the invention, pUCK106.

FIG. 2 shows the DNA sequence of 215-bp inserted at the XbaI site ofpUCK19.

FIG. 3 shows an ethidium bromide staining of polyacrylamide gel of theproduction of an slDNA from pUCK106 and its characteristics.

FIG. 4 shows an autoradiograph of a dried polyacrylamide gel of dimerformation of the slDNA from pUCK106.

FIG. 5 illustrates determination of the DNA sequence of the slDNA frompUCK106.

(A) shows an autoradiograph of a dried polyacrylamide gel of thedetermination of the DNA sequence SEQ ID NO: 19 and SEQ ID NO: 20 of the5' end region of the slDNA.

(B) shows an autoradiograph of a dried polyacrylamide gel of the 5' endsequencing of the loop region SEQ ID NO: 21 of the slDNA.

(C) shows an autoradiograph of a dried polyacrylamide gel of the 3' endsequencing of the loop region SEQ ID NO: 21 of the slDNA.

(D) shows an autoradiograph of a dried polyacrylamide gel of the DNAsequence SEQ ID NO: 22 of the 3' end region of the slDNA.

(E) shows the structure of the slDNA from pUCK106.

FIG. 6 illustrates two possible models of slDNA synthesis.

FIG. 7, lanes 1 and 3 show the HaeIII digest of pBR322 as size markers.Lane 2 shows the slDNA from the preparation from pUC7.

FIG. 8 shows the gene for β-galactosidase in a transformed vector.

FIG. 9 illustrates plasmids pUCK106d.P.O., pUCK106d.P.O.-I, andpUCK106d.P.O.-II.

FIG. 10 illustrates construction of plasmid pXX566.

FIG. 11 shows relation between ampicillin concentration and colonynumber in example 9-3.

FIG. 12 illustrates plasmids pUCKA106d.P.O.,pUCKAGT37, and pUCKACA-37.

FIG. 13 shows DNA sequence inserted into pMS434 and the location of theprimers.

FIG. 14 illustrates construction of plasmids pYES2-8-106, andpYES2-9-106.

FIG. 15 illustrates DNA fragments produced by digestion of pYES2-8-106,pYES2-9-106, and produced slDNA with SalI.

FIG. 16 shows the location of three primers for detecting slDNA.

DEPOSIT OF GENETIC MATERIAL

Plasmid pUCK106 has been deposited with the American Type CultureCollection (ATCC) under Accession No. 68679.

Plasmid pUCK106Δlac^(PO) has been deposited with the ATCC underAccession No. 68680.

EXAMPLES

The following examples are offered by way of illustration and are notintended to limit the invention in any manner. In these examples, allpercentages are by weight if for solids and by volume for liquids, andall temperatures are in degrees Celsius unless otherwise noted.

For convenience and clarity, the Examples refer to and provide also adetailed description of the Figures.

Example 1

FIG. 1 illustrates pUCK19 (circular map), a specific plasmid made asshown below. The open bar in the circular map represents thekanamycin-resistant gene (from Tn 5). The straight open bar shown at theupper right hand side represents a 215-bp DNA fragment inserted at theXbaI site, which contains 35-bp inverted repeat (IR) sequences. Solidarrows show the IR structure. The solid circle design is the origin ofreplication (Ori). The longer open arrow indicates the direction of DNAreplication from the OR. The smaller open arrow shows the position ofthe lac prompter-operator (lac^(PO)).

FIG. 2 shows the DNA sequence of 215-bp inserted at the XbaI site ofpUCK19 consisting of DNAs shown as SEQ ID Nos. 1 and 2 in the sequencelisting respectively. This plasmid is designated herein as pUCK106. Theopen arrows indicate the IR sequences. The HindIII (AAGCTT) site showsthe center of the IR.

Mismatched positions in the IR are shown by two open spaces in thearrows with mismatched bases C and T inserted in the spaces.

The DNA fragment discussed above (which contains the IR) has thefollowing sequence shown as FIG. 2.

Example 2

This example illustrates the production of an slDNA from pUCK106 and itscharacteristics.

(A) E. coli CL83 was transformed with either pUCK19, pUCK106 and withpUCK106Δlac^(PO) and a plasmid DNA fraction was prepared. A DNApreparation (after ribonuclease A treatment) was applied to a 5%acrylamide gel for electrophoresis. The gel was stained with ethidiumbromide. With reference to FIG. 3A, lane 1 shows the HaeIII digest ofpBR322 as size markers; lane 2, the DNA preparation from cells harboringpUCK19; lane 3, pUCK106; and lane 4, pUCK106Δlac^(PO).

pUCK19 is a kanamycin variant of pUC19.

(B) The slDNA from pUCK106 was purified by polyacrylamide gelelectrophoresis followed by various restriction enzyme digestion. Thedigests were analyzed by polyacrylamide (5%) gel electrophoresis, andthe gel was stained by ethidium bromide. With reference to FIG. 3B, lane1, the HaeIII digest of pBR322 as size markers; lane 2, slDNA withoutdigestion; lane 3, slDNA digested with XbaI; lane 4, with HindII; andlane 5 with PvuII.

(C) Heat-denaturation of the slDNA from pUCK106. The purified slDNA (asdescribed above) was solubilized in 10 mM Tris-HCl (pH 8.0) and 1 mMEDTA. The slDNA solution was incubated in a boiling water bath for 3minutes and quickly chilled in an ice bath. Samples were analyzed asdescribed in A. With reference to FIG. 3C, lane 1, the HaeIII digest ofpBR322 as size markers; lane 2, slDNA without heat treatment; and lane3, slDNA heat-denatured followed by quick cooling.

Example 3

This example illustrates dimer formation of the slDNA from pUCK106.

(A) The purified slDNA from pUCK106 as described in FIG. 2 wassolubilized in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl and 10 mM MgCl₂. TheslDNA solution was incubated in a boiling water bath for 3 minutes andthen gradually cooled. The renatured slDNA was digested with XbaI andthe DNA fragments thus generated were labeled at their 5' ends with[γ-³² P]ATP and T4 polynucleotide kinase. These products were applied toa 5% polyacrylamide gel. After electrophoresis, the gel was dried andsubjected to autoradiography.

With reference to FIG. 4A, lane 1, the HaeIII digest of pBR322 as sizemarkers; lane 2, the EcoRI and HindIII digest of γ DNA as size markers.Numbers shown are in base pairs; lane 3, the slDNA from pUCK106 withouttreatment; lane 4, the XbaI digest of the untreated slDNA; lane 5, theslDNA after heat-denaturation followed by gradual cooling; and lane 6,the XbaI digest of the slDNA from lane 5. Bands are marked from "a" to"e" at the right hand side.

(B) Characterization of fragment "d" in FIG. 4A. Fragment "d" purifiedfrom the gel; lane 3, the HindIII digest of the purified fragment "d";and lane 4, the purified fragment "d" was heat-denatured and quicklychilled as described with respect to FIG. 3.

(C) Schematic representation of bands "a" to "e" shown in A and B (FIG.4C). X and H represent XbaI and HindIII sites, respectively. These aretwo other HindIII sites in fragment "a" very close to the XbaI sites(within fragment "c"). These HindIII sites are not shown.

Example 4

This example illustrates determination of the DNA sequence of the slDNAfrom pUCK106.

(A) Determination of the DNA sequence of the 5' end region of the slDNA.0.2 μg of the isolated and purified slDNA was used for sequencing by thechain termination method. Primer "a" (^(5') GGTTATCCACAGAATCAG^(3'))shown as SEQ ID No. 3 in the sequence listing which corresponds to thesequence 96-bp downstream from the origin (see FIG. 5B) was used asprimer.

(B) 5' end sequencing of the loop region of the slDNA. 0.5 μg of theslDNA was digested with SacII and the DNA fragments thus generated werelabeled at the 5' end with [γ-³² P]ATP and T4 polynucleotide kinase. TheDNA fragment migrated at approximately 40-bp was isolated and sequencedby the Maxam-Gilbert method.

(C) 3' end sequencing of the loop region of the slDNA. The SacII digestslDNA was labeled at the 3' end with [γ-³² P]dideoxy ATP using terminaldeoxynucleotidyl transferase. The DNA fragment containing the loopregion was isolated and sequenced by the Maxam-Gilbert method.

(D) DNA sequence of the 3' end region of the slDNA. The slDNA wasdigested with AflIII (see FIG. 5E). The 5' ends were labeled with [γ-³²P]ATP and T4 polynucleotide kinase. The labeled products were separatedby a sequencing gel. The single-stranded DNA which migrated at 76 baseswas isolated and sequenced by Maxam-Gilbert method. By reference to FIG.5D the numbers represent the residue numbers from the origin of pUCK19.

(E) Structure of the slDNA from pUCK106. The slDNA consists of asingle-stranded DNA of 1137 to 1139 bases. The 5' end of the slDNAappears to be heterogeneous; some start from +1 while other start from-1, +2 and +3. The +1 position corresponds to the origin of ColE1 DNAreplication. Thus, various slDNAs have different length 5' endingstrands. At the 3' end a sequence of 16 bases is extended beyond the +1position of the 5' end. The loop is considered to be formed with the 4base sequence (AGCT) corresponding to the sequence at the center of theIR structure, where a HindIII site (AAGCTT) is designed to be placed.The base pair corresponding to the mismatch in the IR structure inpUCK106 was converted from C·T (in pUCK106) to C·G (in the slDNA) isshown between the SacII and PstI sites. The position of primer "a" usedfor DNA sequencing in FIG. 5A is shown by an arrow.

Separation and purification of the slDNAs are performed according tostandard techniques as by following the procedures described inMolecular Cloning, A Laboratory Manual, Sambrook et al., 2d Ed.(Sections 1.121-1.40) ("Sambrook").

Example 5

This example illustrates two possible models of slDNA synthesis (seeFIG. 6). The double-stranded DNA around the origin of the ColE1 DNAreplication is shown on the top. The shaded circle represents the DNAreplication complex which initiates DNA replication from the origin. Theopen arrows on the DNA strand indicate the position of the 35-bpinverted repeat (IR) structure (see FIG. 2) in the DNA sequence. Themismatched base pair (C·T) in the IR structure is also indicated withinthe arrows.

At step 1, the DNA replication fork proceeds from the origin (+1position) to the position indicated by the shaded circle. The newlysynthesized first strand is shown extending from the origin (a solidcircle) to the replication fork. The DNA replication complex reachesimmediately before the mismatched T residue in the IR structure that isshown by solid arrows. At step 2, the 3' end of the nascent stranddetaches from the DNA replication complex and a secondary structure isformed by the IR structure. At step 3, DNA synthesis reinitiates fromthe 3' end of the stem-loop structure utilizing either the nascentstrand (model A) or the upper parental strand (model B) as template. Atstep 4, DNA synthesis proceeds beyond the origin by 16 bases.

In model A, the primer RNA which remains attached at the 5' end of theDNA may be used as the primer RNA. Subsequently, the RNA may be removedresulting in the formation of slDNA. In model B, DNA synthesisterminates at the terH site by a similar mechanism known for thetermination of the second strand DNA synthesis.

It is conceivable that both models A and B can explain the synthesis,and the synthesis may proceed by both routes concurrently, at least forpart of the time. Thus, an appropriate template will be used for thesecond strand other than the strand which was the template for the firststrand.

Example 6 Construction of pUCK106Δlac^(PO)

When the 199-bp PvuII-HincII fragment containing the lacpromoter-operator was deleted from pUCK106 (see FIG. 1), the resultingpUCK106Δlac^(PO) produced a new slDNA which migrated faster than theslDNA from pUCK106 as shown at position (b) in lane 4, FIG. 3A. The sizeof this new slDNA was 360-bp in length, which is shorter than thepUCK106 slDNA by a length nearly identical to the size of the deletionin pUCK106Δlac^(PO).

In FIG. 3A, lane 3 shows the slDNA from the DNA preparation from cellsharboring pUCK106Δlac^(PO).

This experiment supports the model for slDNA synthesis proposed aboveand also indicates that the lac promoter-operator is not essential forslDNA synthesis. This notion was further supported by the fact that theaddition of isopropyl-β-D-thiogalactopyranoside, an inducer of lac, didnot affect the production of the slDNA from pUCK106. However, the reasonfor the reduction of slDNA synthesis from pUCK106Δlac^(PO) is not knownat present.

Example 7

The synthesis of slDNA was not dependent upon the primary sequence ofthe IR structure used for pUCK106. Interestingly, the pUC7 vector byitself, which has an IR structure at the polylinker site is also able toproduce an slDNA corresponding to the DNA fragment from the origin tothe center of the polylinker site.

The isolated and purified slDNA produced from pUC7 was 252-bp in length.A plasmid fraction was prepared and treated as in Example 2 and applied(and stained) to acrylamide gel for electrophoresis.

In FIG. 7, lanes 1 and 3 show the HaeIII digest of pBR322 as sizemarkers.

Lane 2 shows the slDNA from the preparation from pUC7.

The slDNA of pUC7 was amplified by PCR (which is described in furtherdetail herein) and lane 4 (at arrow) shows the slDNA.

Example 8 Confirmation of the slDNA Structure

The slDNA structure and mechanism described above (illustrated in FIG.6) was confirmed as follows.

The DNA fragment that was synthetically constructed was providedintentionally with mismatched bases CT for CG. See FIG. 2 in the openspaces of the IR. After synthesis of the slDNA (with the second strandsnapped-back over the first strand) the mismatch has been repaired. FIG.SE, now CG appears. If the structure were not a snap-back structure, thepolymerase would have read right through the IR, would have read the Tand inserted an A; the new strand would still have contained themismatch. To replace the mismatched T, that IR portion necessarily hadto snap-back onto the first strand, thus allowing the polymerase to usethe first synthesized strand as template to synthesize the second strandand as it synthesizes it, insert the complementary base G in place ofthe mismatched T. This unequivocally establishes the synthesis mechanismand structure of the slDNAs of the invention.

Example 9

This example illustrates regulating gene function by the use of slDNAcontaining antisense sequence, that is, a gene which is reversed inorientation with respect to its promoter (antisense slDNA).

9-1 Construction of plasmids which produce slDNA

pUCK106d.P.O. was prepared from PUCK106 by the deletion of the 199-bpPvuII-HincII fragment containing the lac promotor-operator (FIG. 9).Then, the oligonucleotides with the structures shown as SEQ ID No. 4(antisense) and No. 5 (sense) in the sequence listing were synthesized,annealed, and inserted at the HindIII site of the pUCK106d.P.O.. Theplasmid which produces slDNA containing sense sequence (sense slDNA) wasnamed pUCK106d.P.O.-I and the plasmid which produces antisense slDNA wasnamed pUCK106d.P.O.-II (FIG. 9).

9-2 Preparation of the host E. coli which contains the β-lactamase gene

The EcoRI-HaeII fragment of approximately 1.6-kb containing theβ-lactamase gene from pBR322 (Takara Shuzo) was blunt-ended and insertedat the SmaI site of pHSG399 (Takara Shuzo) The resulting plasmid wasnamed pXX555. The 1695-bp EcoRI-HindIII fragment from pXX555 wasinserted at the EcoRI-HindIII site of the pXX325 derived from miniFplasmid [see Proc. Natl. Acad. Sci. USA, 80, 4784-4788, 1983]. Theresulting plasmid was named pXX566 (FIG. 10). The E. coli MC4100containing the β-lactamase gene was prepared by the transformation withpXX566. The transformant was named MC4100/pXX566.

9-3 Reduction of β-lactamase gene expression by antisense slDNA

E. coli MC4100/pXX566/I and E. coli MC4100/pXX566/II were prepared fromE. coli MC4100/pXX566 by the transformation with pUCK106d.P.O.-I andpUCK106d.P.O.-II, respectively. Each transformant was inoculated intoL-broth containing 50 μg/ml kanamycin and incubated over night at 37° C.The culture was diluted 1:10000 with L-broth and 50 μl of each dilutionwas plated out on agar L-broth plates containing 50 μg/ml kanamycin and50-150 μg/ml ampicillin. After over night incubation at 37° C., thenumber of colonies was counted. Table 1 and FIG. 11 show the relativepercent of the numbers (X-axis: ampicillin concentration, Y-axis: therelative percent, 100% at the concentration of 50 μg/ml ampicillin). Theincrease of relative death rate showed that β-lactamase gene expressionwas reduced by antisense slDNA.

                  TABLE 1                                                         ______________________________________                                        Ampicillin conc.                                                                           MC4100/pXX56/I                                                                            MC4100/pXX566/II                                       (μg/ml) (sense) (antisense)                                              ______________________________________                                        50           100         100                                                    75 83 60                                                                      100 55 22                                                                     150 0 0                                                                     ______________________________________                                    

9-4 Measurement of β-lactamase expression by western blotting

Each single colony of MC4100/pXX566/I and II as in Example 9-3 wasselected at random and inoculated into 5 ml of L-broth containing 20μg/ml ampicillin and 50 μg/ml kanamycin. After over night incubation at37° C., 100 μl of each culture was inoculated into the same fresh media.Cells were harvested at the time when the absorbance at 600 nm reached1.2, washed with 10 mM sodium phosphate buffer (pH 7.1), and resuspendedin 1 ml of the same buffer. The cell density was recorded by measuringthe absorbance at 600 nm. The sonicated lysates of 5.36×10⁸ cells wereelectorophoresed on a 15% SDS-polyacrylamide gel and the separatedproteins were transferred to a piece of PVDF membrane filter (Immobilon,Millipore). The filter was subsequently exposed to rabbitanti-β-lactamase polyclonal antibody (5 Prime-3 Prime, Inc.) anddetection was done with Immno-Stain-Kit (Konica). After treatment withchromogenic substrate, colored bands were appeared at proper position.Those bands indicated that the level of β-lactamase activity in the cellproducing antisense slDNA was 30% lower than that in the cell producingsense slDNA.

Example 10

This example illustrates slDNA capable of forming triple helix (triplehelix forming slDNA).

10-1 Construction of pUCK106Ad.P.O.

The 106-NdeI sequence which consists of the sequences shown as SEQ IDNos. 6 and 7 in the sequence listing was inserted at the NdeI site ofthe pUCK19. The resulting plasmid was named pUCK106A. PlasmidpUCK106Ad.P.O. was constructed by the deletion of 206-bp HincII-VspIfragment containing the lac promoter-operater region from pUCK106A.

10-2 Preparation of the triple helix forming slDNA

The oligonucleotide shown as SEQ ID No. 8 (GT37 sequence) in thesequence listing and the oligonucleotide shown as SEQ ID No. 9 (CA37sequence) in the sequence listing were synthesized, and phosphorylatedat 5' end. The duplexed oligonucleotide was made by annealing with theseoligonucleotides and inserted at the HindIII site of pUCK106Ad.P.O., inthe middle of 106-NdeI sequence. Each plasmid that produces the slDNAcontaining GT37 sequence or CA37 sequence was named pUCKAGT37 orpUCAKCA37, respectively (FIG. 12). E. coli MC4100 were transformed bythese plasmids, and each transformant was named MC4100/pUCKAGT37 orMC4100/pUCKACA37. These transformants and MC4100/pUCK106Ad.P.O.constructed in Example 9-2 were cultured in L-broth containing 70 μg/mlkanamycin. DNA was prepared by alkaline-SDS method, and treated withRNaseA. The slDNA was purified by polyacrylamid gel electrophoresis.

10-3 Preparation of the target DNA

The oligonucleotide shown as SEQ ID No. 10 in the sequence listing andthe oligonucleotide shown as SEQ ID No. 11 in the sequence listing weresynthesized and phosphorylated at 5' end. The duplexed oligonucleotidewas made by annealing with these oligonucleotides and inserted at theXhoI-HindIII site of pMS434 [see Gene, 57, 89-99, (1987)]. This plasmidwas named pMSTR37.

The target DNA of triple helix formation was amplified by PCR withprimer M4 shown as SEQ ID No. 12 in the sequence listing and primer MS1shown as SEQ ID No. 13 in the sequence listing as primers, and pMSTR37as a template.

10-4 Triple helix formation on slDNA

The slDNA prepared in Example 10-2 were labeled with [γ-³² P]ATP byphosphorylation. Triple helix formation was made by mixing radiolabeledslDNA and excess target DNA in a 0.15M NaCl/10 mM MgCl₂ /5 mMtris-acetate (pH7.0) buffer, and then incubated at 37° C. overnight.Triple helix formation was detected by 12% polyacrylamide gelelectrophoresis in 50 mM tris-borate/5 mM MgCl₂ (pH8.3) buffer.

In result, triple helix formation was detected only with the slDNAcontaining GT37 sequence. On the other hand, triple helix formation wasnot detected with either slDNA containing CA37 sequence or intact slDNA.

Example 11

This example illustrates the synthesis of slDNA in Saccharomycescerevisiae.

11-1 Construction of plasmids pYES2-8-106 and pYES2-9-106

The shuttle vector pYES2 (Invitrogen) containing ColE1 and 2μ originswas modified as illustrated in FIG. 14 and the derivatives were used toinvestigate whether slDNA can be produced in yeasts. First, the multiplecloning sites and the Gal I promoter region were removed from pYES2 bydigestion with MluI and SspI. Next, the remaining DNA fragment wasblunt-ended by T4 DNA polymerase and then self-ligated (step 1). Second,the DNA fragment A or B, shown as SEQ ID No. 14 or 15 in the sequencelisting respectively, was inserted at the blunt-ended AvaI site of theplasmid of step 1 (step 2). Finally, the 227-bp BamHI-SalI DNA fragmentfrom pUCK106 was inserted at BamHI-SalI site of the plasmids of step 2(step 3). The resulting pYES2 derivatives were named pYES2-8-106 (leftside in FIG. 14) and pYES2-9-106 (right side in FIG. 14) respectively.

11-2 Purification of plasmid DNA and slDNA from the transformants

Saccharomyces cerevisiae YPH499 cells (Stratagene) were transformed bypYES2-8-106 or pYES2-9-106 by the LiAc procedure [see Journal ofBacteriology, 153, 163-168, (1983)]. These transformants were grown on100 ml of YPD medium (1% yeast extract, 2% bacto-peptone, 2% dextrose)to an OD₆₀₀ of approximately 1.5. The cells were harvested bycentrifugation, and washed once with 10 ml of SCE solution (182 g/lSorbitol, 29.4 g/l Na₂ Citrate, 22.3 g/l Na₂ EDTA). Next, the cells weresuspended in solution I (SCE solution containing 0.1% β-mercaptoethanoland 0.5 mg/ml Zymolyase 100T) and shaken gently at 37° C. for 2 hours togenerate spheroplasts. Next, 8 ml of solution II (0.2 N NaOH, 1% SDS)was added and the suspensions were held on ice for 5 minutes. Next, 6 mlof solution III (60 ml of 5M K-acetate, 11.5 ml of glacial acetic acid,28.5 ml of H₂ O) was added, held on ice for additional 5 minutes, andthen centrifuged. The DNAs were precipitated by addition of isopropanolto the supernatants, washed with 70% ethanol, and resuspended in TEbuffer (10 mM Tris-HCl, 1 mM EDTA, pH7.6). The DNAS were furtherpurified by phenol/chloroform extraction, ethanol precipitation, andusing Qiagen tip 5 (Qiagen Inc.). Finally the purified DNA weresuspended in 20 μl of TE buffer.

11-3 Separation of the slDNA from the plasmid DNA

10 μl of the samples obtained in Example 11-2 were treated with SalI. Bythis process, the plasmid DNA should be changed to 5024-bp linear DNAfragment and slDNA should be divided into two fragments, namely, 127-bpof slDNA and double-stranded linear DNA whose length was unknown (FIG.15). After addition of 2 μg of ColE1-AfaI digest, which was composed of29, 46, 60, 62, 69, 99, 120, 126, 130, 243, 252, 323, 413, 415, 564,778, 950, 976 and 991-bp fragments, the samples were separated on 12%polyacrylamide gel electrophoresis. The lower half of the gel was cutand stained by ethidium bromide, and the gel of the region between120-bp and 130-bp was recovered and sliced. The DNAs were extracted fromthe gel by incubation at 60° C. for 30 min with the addition ofsterilized water. After centrifugation, insoluble impurities wereremoved from supernatant by glasswool column and then the supernatantwas lyophilized. Finally the recoverd DNAs were suspended in 20 μl of TEbuffer.

11-4 Detection of the slDNA by PCR method

The detection of the slDNA in the samples prepared in example 11-3 wascarried out by PCR method. Primer 1871 and primer 20261 shown as SEQ IDNos. 16 and 17 in the sequence listing respectively were prepared todetect the slDNA. Primer 1870 shown as SEQ ID No. 18 in the sequencelisting was prepared to detect the contaminating plasmid DNA (FIG. 16).The efficiencies of amplification with the two combinations of primers,1871/20261 or 1871/1870, were checked by examining the lowestconcentrations of the plasmid DNA which can be detected by PCR. As theresults, the lowest values of the concentrations detectable wereapproximately 2.5×10⁻²⁰ moles of template DNA in the 50 μl reactionmixtures in both cases.

PCR reactions were carried out with 50 μl reaction volumes in which1/100 volumes of PstI digested DNA obtained in the example 11-3 wasincluded as the template DNA. 25 cycles of the following steps wereperformed; at 94° C. for 1 min, at 55° C. for 1 min, and at 72° C. for 1min. After the reactions, 5 μl of the products were analyzed by 6%polyacrylamide gel electrophoresis, stained with ethidium bromide anddetected by FMBIO-100 (Takara Shuzo). In both cases DNAs recovered fromthe cells transformed by pYES2-8-106 or pYES2-9-106, 93 bp fragmentswere amplified with the combination of the primers 1871 and 20261.However no fragments were amplified with primers 1871 and 1870. Thelatter observations showed that the amplified fragments detected in theformer did not derived from the contaminating plasmid DNA but from theslDNA synthesized in the yeast cells.

From these results, it is clearly demonstrated that slDNA can besynthesized in the eucaryotic cells like yeasts. Furthermore, the factthat the transformant containing pYHS2-9-106 produced slDNA suggestedthat slDNA was synthesized by means of not only leading strand synthesisbut also lagging strand synthesis.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS OF THE INVENTION

The inventors have made the fundamental discovery that part of thegenome is directly duplicated from the genome. The mechanism discoveredrequires neither an RNA intermediate nor reverse transcriptase (RT) asis well known. See Weiner et al., Ann. Rev. Biochem., 55, 631 (1986);Kornberg, in DNA Replication (W. H. Freeman and Company, San Francisco,Calif., 1980), pp. 101-166. New and useful DNA structures namedstem-loop DNAs (slDNAs) have been discovered.

By way of introduction, the invention provides several embodiments. Oneembodiment is a method (or process) for synthesizing a novel and usefulssDNA molecule (or structure). Another embodiment is an in vivo and invitro system for synthesizing such molecule which includes a DNAfragment which contains a suitable priming site, an inverted repeat (IR)and other necessary components for synthesizing the slDNAs.

An in vivo system for synthesizing such molecules uses a competentself-replicating vehicle and other components of the system. Anotheraspect of this embodiment is the self-replicating vehicle which containsan inverted repeat, a DNA to serve as template for the replication of afirst strand of the slDNA, a suitable priming site for the template tostart DNA synthesis in the opposite orientation and when needed, thesecond strand of the parent DNA and other components further described.

Another embodiment is the novel ss-slDNA molecules.

The invention provides a method for synthesizing a novel single-strandedDNA (ssDNA) molecule. The molecule comprises a stem-loop structure(slDNA) which stem is constituted of duplexed DNA of annealedcomplementary bases within the ssDNA, which stem forms, at one end, the5' and 3' termini of the ssDNA molecule and at the other end, asingle-stranded loop of DNA joining the opposite ends of the duplexedDNA. The method is carried out in a system which contains theconventional components of DNA synthesis, and the following components:

(a) a template DNA containing a suitable priming site and an invertedrepeat (IR) downstream of said priming site,

(b) a primer for the template to allow the start of DNA polymerization,and

(c) a DNA polymerase to replicate the slDNA from the template.

The method comprises the steps of:

(1) priming the DNA template to allow the start of DNA polymerization,

(2) synthesizing the ssDNA from the primer using one of thedouble-strand of the DNA as template thereby forming one strand,continuing the DNA synthesis of the strand into the IR sequence andallowing the synthesis to cease within the IR sequence,

(3) allowing complementary bases within the newly synthesized strand atthe IR sequence to anneal forming, at one end, a loop of a non-duplexedregion and a duplexed region, the duplexed region functioning as apriming site for continued DNA synthesis,

(4) resuming DNA synthesis using as template the newly synthesizedstrand and/or the other strand of the DNA,

(5) forming the slDNA, and

(6) separating and isolation the slDNA.

The method is carried out in a system which contains all the necessaryconventional components for DNA synthesis. These components may bepresent inherently as when the method is carried out in vivo; they willnormally be introduced into the system when the method is carried out invitro.

The method calls for the presence of a DNA which has a suitable primingsite and an inverted repeat downstream of the priming site. The DNAserves as a template for directing the DNA replication. The primer canbe any oligonucleotide (whether occurring naturally or producedsynthetically) which is capable of acting to initiate synthesis of aprimer extension product which is complementary to a nucleic acidstrand. The method of initiation of DNA claims is of course well known.See Watson, Molecular Biology of the Gene, 3rd Ed., W. A. Benjamin,Inc.; DNA Synthesis: Present and Future, Molineux and Kohiyama, Eds.,(1977) Part V, "G4 and ST-1 DNA Synthesis In Vitro" by Wickner; PartVII, "DNA Synthesis in Permeable Cell Systems from Saccharomycescerevisiae" by Oertel and Goulian. Different polymerases may contributeto the synthesis of the second strand, as opposed to that contributingto the synthesis of the first strand. The primer may be a DNA or RNAprimer. Synthesis is induced in the presence of nucleotides and an agentof polymerization, such as DNA polymerase at a suitable temperature andpH.

The method of the invention uses an agent for polymerization such as anenzyme, which catalyzes the synthesis of the strand along the template.Suitable enzymes for this purpose include, for example, any DNApolymerase like E. coli DNA polymerase I, or Ill, Klenow fragment of E.coli DNA polymerase I, T4 DNA polymerase, T7 DNA polymerase, reversetranscriptase (RT), viral polymerases, including heat-stable enzymes.Any polymerase that will recognize its site of initiation for DNAreplication is appropriate. Generally, when the process is carried outin vivo, these genetic elements will be present; if not or if performedin vitro they will be added to the system.

Any polymerase that will recognize the site of initiation may cause orcontribute to the polymerization While the inventors do not wish to bebound to any particular theory at this time, it is not to be excludedthat the polymerase that contributed to the synthesis of the first DNAstrand also contributes to synthesize the other or second DNA strand.The method thus provides continuing the synthesis of the second stranduntil it terminates at terminus 3' beyond the 5' terminus of the firstformed strand. Thus, there is formed the duplexed stem of the slDNA.

Information on DNA polymerases is available. For instance, see Kornberg,in DNA Replication (W. H. Freeman and Company, San Francisco, Calif.,1980), pp. 101-166, Chapter 4, DNA Polymerase I of E. coli, Chapter 5,Other Procaryotic Polymerases, Chapter 6, Eucaryotic DNA Polymerases andChapter 7.

Other polymerases, such as those that are useful in second-strandsynthesis in cDNA may be considered.

In a specific illustration described above, it is postulated that thepolymerases are two: DNA polymerase III and polymerase I.

When it is desired to replicate a desired or target nucleic acidsequence which is capable of encoding a protein, e.g., a gene as part ofthe synthesized slDNAs, the sequence will be positioned upstream of theIR. For example, when the replication takes place in a replicationvehicle like a vector, e.g., pUCK106, which has an origin of replication(OR), the target nucleic acid sequence will be positioned in between theIR and the OR.

The method of the invention uses a double-stranded DNA fragment whichhas, as described, a priming site and also an inverted repeat (IR),i.e., two copies of an identical sequence present in the reverseorientation. The IR may be present as a sequence or "palindrome".

As will be described hereinafter, certain enzymes will be preferred overothers, such as RT when it is desired to favor introducing random pointmutations in a nucleic acid sequence.

The synthesis of the first strand proceeds in a contiguous manner alongthe dsDNA template through the IR resulting in the replication of theentire plasmid genome. At a certain frequency, the synthesis of the DNAstrand is ceased within the IR, forming a short region of sequence whichis complementary to itself, forms a loop and at the IR sequence annealsto itself. This double-stranded region within the newly synthesizedstrand is recognized as the priming site for the second or other DNAstrand. Synthesis of the second strand starts using the first formedstrand and/or the other parent strand of the DNA as template. Thus,template switching is believed to occur.

As the second strand is synthesized by the polymerase incorporating thenucleotides, the stem is formed of the annealed complementary basesresulting in its duplexed structure with internal complementarity.

The synthesis of the second strand proceeds all the way past the firstnucleotide of the first synthesized strand through the RNA primer ofthis first strand which eventually degrades, thus providing a 3'overhang. In one specific illustration, the DNA synthesis is believed toterminate at the terH site by a mechanism similar as described inDasgupta et al., Cell 51, 1113 (1987).

By appropriate manipulations the length of the 3' end overhang can becontrolled, e.g., lengthened, as by moving the terH site downstream fromthe priming site.

Further, instead of a stem with a 3' end overhang, it is consideredfeasible to block the synthesis of the second strand before the end ofthe first strand by placing an appropriate termination site, e.g., terHupstream of the priming site. Thus, it is contemplated that eitherstrand can be longer by a predetermined length with respect to theother.

As the synthesis of the second strand ceases, the template and theformed slDNA separate.

The method of the invention may be repeated in cycles as often as isdesired. If any of the necessary components are becoming depleted, theyare resupplied as it becomes necessary.

When the method is carried out in vivo there is provided a suitablecompetent replicating vehicle which carries the necessary template DNAfragment having a priming site and an IR downstream of the priming site.The DNA fragment carrying the IR will normally be inserted into arestriction site unique in a polylinker sequence. A plasmid has beenused in which the polylinker has an IR (and symmetrical restrictionsites). When not inherently present, the DNA fragment will be providedwith a primer to the DNA sequence; the polymerase may be indigenous tothe vehicle or not. The vehicle will contain all other necessaryelements for replication and forming the slDNAs.

From the foregoing it will be understood that any self-replicatingvector which contains a DNA fragment to serve as template, an IRsequence, the necessary elements to prime and continue the replicationof the strand that will form the slDNA, is suitable to synthesize theslDNAs.

Another major embodiment of the invention provides the new ss-slDNAs.These structures have already been described hereinabove. Additionaldescription is provided hereinafter.

The new structure which has been named "stem-loop" or "slDNA" issingle-stranded. An illustration of an slDNA is shown in FIG. 5E (frompUCK106) and FIG. 7 (from pUC7).

Typical slDNAs contain a duplexed double-stranded stem of annealedcomplementary bases and a loop of a single-strand of nucleotidesconnecting the two strands of the steam. The single-strandedness of theloop is another interesting feature of the slDNAs of the invention.slDNAs can include loop of a very short sequence of nucleotides orconsiderably longer ones. The slDNAs may contain a nucleotide sequencejust long enough to form the loop and base pairing forming a shortduplexed double-strand. The minimum size should allow for base pairingenough to provide for priming site for the start of synthesis of thesecond strand. The minimum size of the loop may be limited by thestrains on the bases that would prevent their pairing into a stablestructure. The maximum size is influenced by the use intended for theslDNAs. The loop illustrated in FIG. 5B is constituted of four bases;loops of 10, 20 or more bases can be conceived. The single-strandednessof the loop of the slDNAs is a feature which may be quite useful in theutilities proposed for the slDNAs.

Yet another interesting structure contemplated for the slDNAs is thedouble slDNAs. In this structure, the free ends of the single-strands oftwo slDNAs are ligated. The structure is expected to be extremelystable. On exposure to conditions which normally denature DNAs, thesessDNAs are likely to "snap-back" to their original structure. Joining ofthe strands will be carried out by conventional procedures with DNAand/or RNA ligases. Such structures can also carry selected genes forencoding proteins and their thus provide interesting new practicalpossibilities.

It is believed that the stability, an important property of the slDNAs,tends to increase with longer duplexed tails; thus, such structures arefavored when this is a property which is to be emphasized. It should benoted that all slDNAs generated from a single replicating vehicle arenot necessarily identical in size. In the illustration shown above,slDNA from pUCK106, the 5' end of the first strand appears to beheterogeneous, some strands starting front the +1 position (whichcorresponds to the origin of ColE1 replication) (see Tomizawa et al.,Proc. Natl. Acad. Sci. USA, 74, 1865 (1977)), while other strands startfrom -1, +2 and +3. Thus, the DNAs may be considered as family ofanalogous slDNAs.

It may be noted that the presence of one or more mismatch in the DNAfragment beyond the IRs does not adversely affect the synthesis nor thestructure of the slDNAs. This is illustrated by the mismatch T for G, inthis case 25 nucleotides away from the center of the palindrome (seeFIG. 2). This mismatch was repaired in the synthesis of the slDNA.

Several utilities for the slDNAs of the invention are proposed hereinwhich take advantage of the ssDNA overhang of one end of the tail overthe other. It will therefore be appreciated that this is an importantfeature of the new structures of the invention.

The synthesis of the slDNAs in the illustrated plasmid is descriptive ofa best mode of the invention. However, any vector which contains the IRand other components described herein is suitable for synthesizing theslDNAs.

IR is a structure frequently by occurring in procaryotes and ineucaryotes. Any such IR may be used in the invention. IR sequences mayalso be prepared synthetically. An illustration is the IR synthesizedand shown in FIG. 2

Sequences of IR or palindrome sequences have been obtained from E. coli(Gilson et al., Nucl. Acids. Res., 18, 3941 (1990)); Gilson et al.,Nucl. Acids Res. 19, 1375 (1991) reported palindromic sequences from E.coli and Salmonella enteritica (a palindromic unit sequence 40nucleotides long). Chalker et al., Gene, 71, (1):201-5 (1988) reportsthe propagation of a 571-bp palindrome in E. coli; inverted repeats arereported by Lewis et al., J. Mol. Biol. (England), 215, (1):73-84 (1990)in Bacillus subtilis (a 26-base pair repeat), and Saurin, Comput. Appl.Biosci., 3, (2):121-7 (1987) discusses the use of a new computer programto search systematically for repetitive palindromic structures in E.coli. The following U.S. patents disclose palindromic sequences: U.S.Pat. Nos. 4,975,376; 4,863,858; 4,840,901; 4,746,609; 4,719,179;4,693,980 and 4,693,979.

Palindromes have been defined to include inverted repetitious sequencesin which almost the same (not necessarily the same) sequences run inopposite direction. Though some are short (3-10 bases in one direction),others are much longer, comprising hundreds of base pairs. Watson,Molecular Biology of the Gene, 3rd Ed., pps. 224-225.

The IR in the DNA fragment can vary considerably in size. Withoutintending to be limited slDNAs with inverted repeats of 10 to 30 orlonger nucleotides may be considered. Inverted repeats have beenreported to contain more than 300-bp. Current Protocols, Section 1.4.10.

The slDNAs can be the synthesis product of procaryotic or eucaryotichost expression (e.g., bacterial, yeast and mammalian cells).

Examples of appropriate vectors such as a plasmid and a host celltransformed thereby are well known to one skilled in the art. Amongappropriate hosts for plasmid carrying the necessary components areprocaryotes and eucaryotes. Procaryotes include such microorganisms asthose of the genus Escherichia, in particular E. coli; of the genusBacillus, in particular, B. subtilis. Eucaryotes include such as yeast,animal, and plant, and also include such microorganim as animal cellsand plant cells.

Plasmids capable of transforming E. coli include for example, the pUCand the ColE1 type plasmids. See U.S. Pat. No. 4,910,141. Plasmidscapable of transforming E. coli include for example, ColE1 type plasmidsin general. Other appropriate plasmids for transforming E. coli include:pSC101, pSF2124, pMB8, pMB9, pACYC184, pACYC177, pCK1, R6K, pBR312,pBR313, pML2, pML21, ColE1AP, RSF1010, pVH51, and pVH153.

Plasmids capable of transforming B. subtilis include: pC194, pC221,pC223, pUB112, pT127, pE194, pUB110, pSA0501, pSA2100, pTP4, pTP5 andtheir derivatives. Plasmids capable of transforming both B. subtilis andE. coli are described in J. Bacteriol., 145, 422-428 (1982); Proc. Natl.Acad. Sci. USA, 75, 1433-1436 (1978) and Principles of Gene Manipulation2nd Ed., Carr et al. Eds., University of Ca. Press, Berkeley, 1981, p.48.

Of special interest for carrying out the synthesis of the slDNAs ineucaryotes are plasmids capable of transforming S. cerevisiae: pMP78,YEp13, pBTI1, pLC544, YEp2, YRp17, pRB8 (YIp30), pBTI7, pBTI9, pBTI10,pAC1, pSLe1, pJDB219, pDB248 and YRp7. Also to be considered are YIp5,pUC-URA3, pUC-LEU2 and pUC-HIS3. See page 285 and pages 373-378 inMethods in Enzymology, Vol. 194, "Guide to Yeast Genetics and MolecularBiology", edited by Guthrie and Fink (1991), Academic Press, Inc. Otheryeast vectors are described at pages 100-104 in ExperimentalManipulation of Gene Expression, edited by Masayori Inouye, AcademicPress, Inc. (1983).

Further, of particular interest are shuttle vectors which can be used totransform E. coli as well as yeast like S. cerevisiae. Such vectorsinclude the following: pKB42 and pYC1. Other examples are listed in thesection on "Cosmid Vectors for Low and Higher Eucaryotes" in A PracticalGuide to Molecular Cloning, 2nd Edition by Bernard Perbal (1988), Wileyand Sons. Other suitable vectors are described in Vol. 2, Sections13.4.1, 13.4.2 (1989), Current Protocols. Other suitable vehiclesinclude such popular multicopy vectors like YEp24 (Botstein et al.,Gene, 8, 17 (1979)) and pJDB207 (Beggs, Genetic Engineering (Ed.Williamson), Vol. 2, p. 175, Academic Press (1982)). Others that may beselected include plasmids of the classes YEp, like YEp51, YEp52 andpYES2.

Examples of commercially available eucaryotic vectors for carrying outthe present invention are pSVL and pKSV-10 in for example, COS, CHO andHeLa cells. Other examples are listed in A Practical Guide to MolecularCloning.

Culturing and fermentation of the transformed hosts is carried out bystandard and conventional methods known in the art. See for example,Methods in Enzymology, Vol. 185, Gene Expression Technology (Goeddel,editor) 1990 (in particular, Growth of Cell Lines); for yeasts, seeMethods in Enzymology, Vol. 194, Guide to Yeast Genetics & MolecularBiology; growth conditions for E. coli are described in CurrentProtocols in Molecular Biology, Vol. 1, at pages 1.1.1, 1.1.2, 1.1.3,1.1.4 and 1.3.1 and Molecular Cloning: A Laboratory Manual, 2nd Editionat page 1.21 and purification of plasmid DNA is described at page 1.23,culturing growth conditions suitable for mammalian cells are describedin Current Protocols, Vols. 1 and 2 at pages 9.0.4-9.0.6, 9.1.1, 9.1.3,9.2.5, 9.4.3, 11.5.2, 11.6.2 and 11.7.3.

In summary, when the necessary components for initiating and thesynthesis of the single first strand are supplied in a replicatingvehicle, namely, a DNA template fragment with an initiation site, and anIR (and the polymerase(s)), a slDNA is expected to be formed.

When the synthesis of the slDNAs of the invention is performed in vitro,the synthesis will be performed in a medium which includes conventionalcomponents for the synthesis of nucleotide strands using the DNAfragment as template. Generally, the synthesis will take place in abuffered aqueous solution, preferably at a pH of 7-9. Preferably, amolar excess of the oligonucleotides over the DNA template strand. Thedeoxyribonucleoside triphosphates dATP, dCTP, dGTP and TTP are also acomponent of the synthesis mixture. Heating and then cooling of thesolution is performed. The polymerase is then added and the synthesisproceeds. These components are called "conventional components" for thepurpose of the invention described herein.

Oligonucleotide synthesis may be carried out by a number of methodsincluding those disclosed in U.S. Pat. No. 4,415,734, and in Matteuci etal., J. Am. Chem. Soc., 103 (11):3185-3191 (1981), Adams et al., J. Am.Chem. Soc., 105 (3):661-663 (1983), and Bemcage et al., TetrahedronLetters, 22 (20):1859-1867 (1981).

The methods of the present invention make use of techniques or geneticengineering and molecular cloning. General techniques of geneticengineering and molecular cloning are included in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory, 1990, A Practical Guide to Molecular Cloning, 2nd Ed.,Bernard Perbal (1988), Methods in Enzymology, Volume 68, Recombinant DNA(Wu, editor), Academic Press, N.Y., 1979, Methods in Enzymology, Volume185, Gene Expression Technology (Goeddel, editor) 1990, CurrentProtocols in Molecular Biology, Vols. 1, 2 and 3.

The slDNAs of the invention have several interesting utilities. Themethod of the invention may be used to provide random mutations in aselected gene. Such a system is illustrated in FIG. 8 which shows thegene for β-galactosidase in a transformed vector.

The method comprises synthesizing an slDNA containing a gene of interestlocated in the stem, isolating the slDNA, cutting out the gene from theslDNA, cloning it into an appropriate replicating vehicle and expressingthe protein encoded by the gene. The proteins can be tested for thedesired activity. By standard procedures the colonies can be screened toidentify those with mutated genes.

An illustration for generating and identifying mutations proceeds asfollows. Into pUCK19 (See Example 1) which harbors the lacZ gene, thereis ligated the 215-bp DNA fragment consisting of DNAs shown as SEQ IDNos. 1 and 2 in the sequence listing respectively, containing the 35-bpIR (described in Example 1) between the DNA fragment and the OR (asshown in Example 1). The plasmid is transformed into competent E. coliCL83 which is grown under standard conditions. Thereafter, the lacZ geneis isolated and inserted into pUCK19 (without the IR-containingfragment). pUCK19 is digested with KasI and EcoRI and the isolating lacZgene is transformed into E. coli CL83. The frequency of mutation isscored by the known in vivo test using β-gal, a colorless substrate,which is hydrolyzed to give a dark blue product. When the colonies arecolorless, this indicates that no β-galactosidase has been produced.

Other genes of lac gene family, e.g., lacY or lacA, or other suitablegenes can be used for this screening test. The gene encoding the protein(or polypeptide) of interest may also be used to determine the frequencyof mutations and selection of appropriate gene encoding the derivedprotein (or polypeptide).

This screening procedure permits selection of competent vectors otherpolymerases that are more likely to introduce or increase the rate ofmutation in the target gene. Thus, a selected gene such as the lacZ genecan be used as the screening gene. Reverse transcriptase known to haveless replication fidelity, appears to be an enzyme of choice for thepurpose introducing mutations in a target gene encoding a desiredprotein.

The desired target protein(s) is then expressed by a competent selectedtransformed host carrying the mutated gene and selected for its desiredbiological properties.

The frequency of mutation is believed to be influenceable by selectingappropriate enzymes that are known to have less fidelity in replication.Thus, when target DNA fragments or genes are amplified, the systemdepends on the degree of replication fidelity or the infidelity, thatis, the frequency of error made by the DNA polymerases in replicatingthe inserted DNA fragment or gene. Thus for each replication error,random mutations are introduced into the genes. The higher the degree ofinfidelity of the DNA polymerase, the greater the number of mutatedgenes, and vice versa.

In one above-illustrated embodiment of the invention in which it isbelieved the PolIII and polI were active, it is expected that the formerhas less fidelity, since PolI is known to have a self-correctingfunction. Thus, the replicating fidelity of the system can be regulatedby appropriate selection of the DNA polymerases. Generally, it isbelieved that random mutations are more likely to be introduced by thepolymerase synthesizing the second strand. An interesting candidatewould be RT.

Genes carrying the desired mutation(s) are useful in chromosomalcross-over. By this method the genes of interest or the slDNA carryingthe mutated gene of interest can be made to integrate and exchangegenetic information from one similar molecule to another. The mutatedgene locates within the genome a sequence that is similar to the vectorsequence and the homologous gene is replicated by the mutated gene.

In this manner there can be generated new strains of microorganisms (orhosts) that contain the mutated gene and will express a desired protein.The slDNA carrying the mutated gene can be made in vitro or in vivo.

The polymerase chain reaction (PCR) is a rapid procedure for in vitroenzymatic amplification of a specific segment of DNA. The standard PCRmethod requires a segment of double-stranded DNA to be amplified, andalways two-single stranded oligonucleotide primers flanking the segment,a DNA polymerase, appropriate deoxyribonucleoside triphosphate (dNTPs),a buffer, and salts. See Current Protocols, Section 15.

The ss-slDNA of the invention can be amplified with a single primer.This feature considerably simplifies the amplification, helps toovercome the problem of one primer finding its proper initiation site,renders it more economical and helps overcome problems associated withthe traditional PCR method.

The amplification of the slDNA comprises denaturing the slDNA to form asingle-stranded DNA (from 3' to 5' ends). The reaction follows the usualsequence: priming from 3' end, polymerase reaction, denaturing andannealing. It is carried out for 25 cycles leading to million-foldamplification of slDNA. The slDNA can be carrying a gene for encoding atarget protein.

A recent report in Science 252, 1643-1650 (Jun. 21, 1991) entitled"Recent Advances in the Polymerase Chain Reaction" by Erlich et al.discusses problems associated with primers and improvements that arebeing proposed to the PCR method.

Accordingly, a method for amplification of the ss-slDNA structures ofthe invention by a method using one primer is of great interest. TheslDNA could be carrying a gene of interest, such as a mutated genehaving improved biological properties.

The in vivo production of the slDNAs of the invention can be somanipulated to provide a desired sequence. The slDNAs so produced maythen be used as antisense DNA.

A fascinating utility that is being considered is the role that slDNAsof the invention can play on the formation of triple helix DNA, ortriplex DNA, and the resulting new triplex slDNA structures. A recentreport in Science, 252, 1374-1375 (Jun. 27, 1991), "Triplex DNA FinallyComes of Age", highlights the timeliness of the present invention.Triplex DNA can be formed by binding a third strand to specificrecognized sites on chromosomal DNA. Synthetic strands of sizespreferably containing the full complement of bases (such as 11-15 andhigher), are discussed. The slDNAs of the invention with long 3' (or 5')ends (and the loop of non-duplexed bases) would appear to be excellentcandidates. The resulting triplex DNA is expected to have increasedstability and usefulness. New therapies based on the triple helixformation, including in AIDS therapy and selective gene inhibition andothers are proposed in the Report.

slDNA containing such DNA sequence regulating gene function as antisensesequence and sequence with the ability of triple helix formation(hereinafter referred to "antigene") can be produced and used in vivo asDNA molecule regulating gene function.

It is the most effective method for the regulation of the specific geneexpression to act on the gene directly because life phenomenon iscontrolled under the information from the genome.

There are some methods for the regulation of the gene expression, forexample, antisense RNA, antisense DNA, and the DNA with the ability oftriple helix formation. As for antisense RNA or antisense DNA, theyanneal the target mRNA because they are complement to the target gene,and they inhibit the translation from mRNA to protein by double strandedformation. Antisense RNA can be produced in a cell by some promoter, butit is difficult to produce antisense DNA in vivo because of its singlestranded DNA. So we are obliged to supply the synthetic DNA as antisenseDNA into the cell and regulate gene expression.

As for the DNA with the triple helix formation, the third singlestranded DNA is bind to double stranded DNA which has poly purine (G orA) at one strand and poly pyrimidine (C or T) at the other strand byHoogsteen binding activity. This third single stranded DNA has theability of triple helix formation, and its many applications are offeredfor not only the gene regulation but also the fields of gene technology.Like antisense DNA, in vivo production of single stranded DNA with theability of triple helix formation is very difficult and therefore we areobliged to supply the synthetic DNA into the cell and regulate geneexpression. In vitro production of antisense DNA or DNA with the abilityof triple helix formation are easy by DNA synthesizer, however, it isnot known of the artificial production of single stranded DNA in vivoexcept msDNA. But msDNA is a single stranded cDNA transcribed form mRNAby reverse transcriptase in a cell and therefore its application is verydifficult because of its complex synthetic process. On the other hand,the slDNA with the sequence for gene regulation can be produced in vivowithout mRNA intermediate and be applied to gene regulation.

When there is inverted repeat sequence (IR sequence) on the way to thedirection of gene duplication, the first strand DNA synthesis anneal toanother template DNA at IR sequence, and then the second strand DNAsynthesis begins. The DNA synthesis proceeds to the origin of DNAduplication and it is terminated at the terminate site of transcription.And finaly this single stranded DNA separated as slDNA from the originaltemplate DNA. Loop region and 3' end or 5' end of slDNA form singlestrand. So it is possible for the antisense DNA or DNA with the abilityof triple helix formation to be inserted into these single strandedregion. slDNA with anti sense DNA sequence anneals the target RNA toinhibit the gene function, and further more the target RNA is cleaved bythe cellular RNase H activity. As slDNA with the ability of triple helixformation is also produced at gene duplication, it is always produced invivo and it enables the regulation of the target double stranded DNA.The method for insertion of antisense DNA or DNA with the ability oftriple helix formation into slDNA are described in detail.

To insert antigene DNA into slDNA, in the case of using plasmid, IRsequence should be on the way to the direction of plasmid duplicationand the antigene sequence should be cloned between two IR sequence. Andafter the transformation, the cell containing this plasmid produceantigene slDNA continuously at the plasmid duplication. slDNA has overhang structure at 5' end or 3' end because in the process of slDNAsynthesis 5' end priming site of slDNA is different from 3' end terminalsite. Therefore it is possible to insert antigene into these over hangregions of slDNA. To produce the slDNA which contains antigene at 3' endor 5' end, antigene sequence should be cloned between the priming siteand terminate site of slDNA from the plasmed with IR sequence. And aftertransformation, single stranded DNA with antigene at 5' end or 3' end ofslDNA is produced.

Originaly slDNA was discovered in E. coli as procaryote, and this slDNAcan be also expressed in yeast as eucaryote. So slDNA is useful forantigene production and gene regulation in eucaryote, such as mammalian.

It can be seen that the present invention is providing a significantcontribution to arts and science.

REFERENCES

1. Weiner et al., Ann. Rev. Biochem., 55, 631 (1986)

2. Inouye and Inouye, TIBS, 16, 18 (1991a)

3. Inouye and Inouye, Ann. Rev. Microbiol., 45, 163 (1991b).

4. Higgins et al., Nature, 298, 760 (1982)

5. Gilson et al., EMBO. J., 3, 1417 (1984)

6. Gilson et al., Nucl. Acids Res., 19, 1375 (1991)

7. Gilson et al., Nucl. Acids Res., 18, 3941 (1990)

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9. Tomizawa et al., Proc. Natl. Acad. Sci. USA, 74, 1865 (1977)

10. Molecular Cloning, A Laboratory Manual, Sambrook et al., 2d Ed.(Sections 1.121-1.40)

11. Kornberg, in DNA Replication (W. H. Freeman and Company, SanFrancisco, Calif., 1980), pp. 101-166, and Chapter 4, DNA Polymerase Iof E. coli, Chapter 5, Other Procaryotic Polymerases, Chapter 6,Eucaryotic DNA Polymerases and Chapter 7

12. Watson, Molecular Biology of the Gene, 3rd Ed., W. A. Benjamin, Inc.

13. DNA Synthesis: Present and Future, Molineux and Kohiyama, Eds.,(1977) Part V, "G4 and ST-1 DNA Synthesis In Vitro" by Wickner; and PartVII, "DNA Synthesis in Permeable Cell Systems from Saccharomycescerevisiae" by Oertel and Goulian

14. Dasgupta et al., Cell, 51, 1113 (1987)

15. Chalker et al., Gene, 71, (1):201-5 (1988)

16. Lewis et al., J. Mol. Biol. (England), 215, (1):73-84 (1990)

17. Saurin, W., Comput. Appl. Biosci., 3, (2):121-7 (1987)

18. U.S. Pat. Nos. 4,975,376; 4,863,858; 4,840,901; 4,746,609;4,719,179; 4,693,980 and 4,693,979

19. Current Protocols, Section 1.4.10

20. U.S. Pat. No. 4,910,141

21. J. Bacteriol., 145, 422-428 (1982)

22. Proc. Natl. Acad. Sci. USA, 75, 1433-1436 (1978)

23. Principles of Gene Manipulation 2nd ed., Carr et al. Eds.,University of Ca. Press, Berkeley, 1981, p. 48

24. Methods in Enzymology, Vol. 194, "Guide to Yeast Genetics andMolecular Biology", edited by Guthrie and Fink (1991), Academic Press,Inc., pps. 285 and 373-378

25. Experimental Manipulation of Gene Expression, edited by MasayoriInouye, Academic Press, Inc. (1983), pps. 100-104

26. A Practical Guide to Molecular Cloning, "Cosmid Vectors for Low andHigher Eucaryotes", 2nd Edition by Bernard Perbal (1988), Wiley and Sons

27. Current Protocols, Vol. 2, Sections 13.4.1, 13.4.2 (1989)

28. Botstein et al., Gene, 8, 17 (1979)

29. Beggs, Genetic Engineering (ed. Williamson), Vol. 2, p. 175,Academic Press (1982)

30. Methods in Enzymology, Vol. 185, Gene Expression Technology(Goeddel, editor) 1990 (in particular, Growth of Cell Lines)

31. Current Protocols in Molecular Biology, Vol. 1, at pages 1.1.1,1.1.2, 1.1.3, 1.1.4 and 1.3.1

32. Current Protocols, Vols. 1 and 2 at pages 9.0.4-9.0.6, 9.1.1, 9.1.3,9.2.5, 9.4.3, 11.5.2, 11.6.2 and 11.7.3

33. U.S. Pat. No. 4,415,734

34. Matteuci et al., J. Am. Chem. Soc., 103 (11):3185-3191 (1981)

35. Adams et al., J. Am. Chem. Soc., 105 (3):661-663 (1983)

36. Bemcage et al., Tetrahedron Letters, 22 (20):1859-1867 (1981)

37. Methods in Enzymology, Volume 68, Recombinant DNA (Wu, R., editor),Academic Press, N.Y., 1979

38. Current Protocols in Molecular Biology, Vols. 1, 2 and 3

39. Current Protocols, Section 15

40. Science, 252, 1643-1650 (Jun. 21, 1991) entitled "Recent Advances inthe Polymerase Chain Reaction" by Erlich et al.

41. Science, 252 1374-1375 (Jun. 27, 1991), "Triplex DNA Finally Comesof Age"

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 22                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 215 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - CTAGAGATAT GTTCATAAAC ACGCATGTAG GCAGATAGAT CTTTGGTTGT GA -            #ATCGCAAC     60                                                                 - - CAGTGGCCTT ATGGCAGGAG CCGCGGATCA CCTACCATCC CTAATGACCT GC -            #AGGCATGC    120                                                                 - - AAGCTTGCAT GCCTGCAGGT CATTAGGTAC GGCAGGTGTG CTCGAGGCGA AG -            #GAGTGCCT    180                                                                 - - GCATGCGTTT CTCCTTGGCT TTTTTCCTCT GGGAT       - #                       - #      215                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 215 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - CTAGATCCCA GAGGAAAAAA GCCAAGGAGA AACGCATGCA GGCACTCCTT CG -            #CCTCGAGC     60                                                                 - - ACACCTGCCG TACCTAATGA CCTGCAGGCA TGCAAGCTTG CATGCCTGCA GG -            #TCATTAGG    120                                                                 - - GATGGTAGGT GATCCGCGGC TCCTGCCATA AGGCCACTGG TTGCGATTCA CA -            #ACCAAAGA    180                                                                 - - TCTATCTGCC TACATGCGTG TTTATGAACA TATCT       - #                       - #      215                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GGTTATCCAC AGAATCAG             - #                  - #                      - #  18                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 215 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - TATGGATATG TTCATAAACA CGCATGTAGG CAGATAGATC TTTGGTTGTG AA -             #TCGCAACC     60                                                                 - - AGTGGCCTTA TGGCAGGAGC CGCGGATCAC CTACCATCCC TAATGACCTG CA -            #GGCATGCA    120                                                                 - - AGCTTGCATG CCTGCAGGTC ATTAGGTACG GCAGGTGTGC TCGAGGCGAA GG -            #AGTGCCTG    180                                                                 - - CATGCGTTTC TCCTTGGCTT TTTTCCTCTG GGACA       - #                       - #      215                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 215 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - TATGTCCCAG AGGAAAAAAG CCAAGGAGAA ACGCATGCAG GCACTCCTTC GC -            #CTCGAGCA     60                                                                 - - CACCTGCCGT ACCTAATGAC CTGCAGGCAT GCAAGCTTGC ATGCCTGCAG GT -            #CATTAGGG    120                                                                 - - ATGGTAGGTG ATCCGCGGCT CCTGCCATAA GGCCACTGGT TGCGATTCAC AA -            #CCAAAGAT    180                                                                 - - CTATCTGCCT ACATGCGTGT TTATGAACAT ATCCA       - #                       - #      215                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - AGCTTACTAG TCATACTCTT CCTTTTTCAA TGCTAGCA      - #                      - #     38                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - AGCTTGCTAG CATTGAAAAA GGAAGAGTAT GACTAGTA      - #                      - #     38                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - AGCTTTTGGT GGGTGGGTGG GTGGGTGTTG TGTGGGTGGG TGGGTTTTA  - #                   49                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - AGCTTAAAAC CCACCCACCC ACACAACACC CACCCACCCA CCCACCAAA  - #                   49                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 62 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - TCGAGGCCTC CCTCCCTCCC TCCCTCTTGA CACCCTCCCT CCCATTTGTT AT -             #AATGTGTG     60                                                                 - - GA                  - #                  - #                  - #                  62                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 62 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - AGCTTCCACA CATTATAACA AATGGGAGGG AGGGTGTCAA GAGGGAGGGA GG -             #GAGGGAGG     60                                                                 - - CC                  - #                  - #                  - #                  62                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - ATCCTGATGC CTGCTCTGCG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - GTTTTCCCAG TCACGAC             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 195 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - TGGCCAGAGA GAGAAAGAGA AGAAGAAAAG ATCTTAGCAT ACGATTTAGG TG -             #ACACTATA     60                                                                 - - GAATACACGA ATTCGAGCTC GGTACCCGGG GATCCTCTAG AGTCGACCTG CA -            #GGCATGCA    120                                                                 - - AGCTTGCGGC CGCATCCCTA TAGTGAGTCG TATTACGATG GGCCCTCCCT CC -            #TCTCCCCT    180                                                                 - - CCTCCCTCGA GGCCT              - #                  - #                      - #   195                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 195 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - TGGCCAGAGA GAGAAAGAGA AGAAGAAAAG ATCTTAGCAT ACGATTTAGG TG -             #ACACTATA     60                                                                 - - GAATACACAA GCTTGCATGC CTGCAGGTCG ACTCTAGAGG ATCCCCGGGT AC -            #CGAGCTCG    120                                                                 - - AATTCGCGGC CGCATCCCTA TAGTGAGTCG TATTACGATG GGCCCTCCCT CC -            #TCTCCCCT    180                                                                 - - CCTCCCTCGA GGCCT              - #                  - #                      - #   195                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - GGTCTAGATC CCAGAGGAAA AAAG          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - TGATCCGCGG CTCCTGCCAT AAGG          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - GGTCTAGAGA TATGTTCATA AAC           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - AGGCCGCGTT GCTGGCGTTT            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - AAACGCCAGC AACGCGGCCT            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - ACCATCCCTA ATGACCTGCA GGCATGCAAG CTTGCATGCC TGCAGGTCAT TA -             #GGGATGGT     60                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - CCGGCGCAAC GACCGCAAAA AGGTATCCGA GGCG       - #                  -     #        34                                                                   __________________________________________________________________________

We claim:
 1. A method for synthesizing an isolated stem-loop DNAconstruct (slDNA), wherein the slDNA construct comprises a single strandof DNA containing an inverted repeat having a secondary structure thatcomprises a single-stranded loop and a double-stranded stem having asingle 5' terminus and a single 3' terminus and a DNA antigene sequencepositioned in the loop of the construct, or in a single-strandedsequence overhanging 5' to the inverted repeat, wherein the method isperformedin an environment that contains the necessary components forDNA polymerase activity and a DNA template containing a priming site andan inverted repeat (IR) downstream of said priming site, and a DNAantigene fragment positioned between said priming site and the IR, orbetween the inverted repeat sequences, a primer to start DNApolymerization, and a DNA polymerase, wherein said method comprises thesteps of:priming the template to start DNA polymerization, synthesizinga single stranded DNA (ssDNA) by starting from the primer forming asingle-stranded DNA using one of the double-strands of the DNA templateas the parental strand, continuing the DNA synthesis into the invertedrepeat sequence, forming a short origin of sequence which iscomplementary to and anneals to itself, thereby forming another primingsite, continuing DNA synthesis using as the template the newlysynthesized strand or the other parental strand of the double-strand DNAor both, thereby forming the single-stranded slDNA, and isolating saidslDNA.
 2. The method of claim 1 wherein the slDNA that is formed has a5' overhang.
 3. The method of claim 1 wherein the slDNA that is formedhas a 3' overhang.
 4. The method of synthesis of claim 1 which iscarried out in vivo.
 5. The method of synthesis of claim 1 which iscarried out in vitro.
 6. The method of claim 4 wherein the synthesis isin a prokaryotic or eukaryotic cell.
 7. The method of claim 1 whereinthe vector is a plasmid.
 8. The method of claim 7 wherein the plasmid isan E. Coli plasmid.
 9. The method of claim 8 wherein the plasmid ispUCK106.
 10. The method of claim 6 wherein the prokaryotic cell is E.coli or B. subtilis.
 11. The method of claim 6 wherein the eukaryoticcell is from a mammal, a plant, or a yeast.
 12. The method of claim 11wherein the yeast is a Saccharomyces yeast.
 13. The method of claim 12wherein the yeast is Saccharomyces cerevesiae.
 14. The method of claim 1wherein the antigene fragment is positioned between the inverted repeatsequences.
 15. The method of claim 1 wherein the antigene fragment ispositioned between the priming site and the inverted repeat.
 16. Themethod of claim 1 wherein there are generated a family of slDNAs ofdifferent sizes.
 17. The method of claim 6 wherein the slDNA isisolated.
 18. The method of claim 17 wherein the slDNA is purified. 19.The method of claim 6 wherein the antigene sequence is selected from thegroup consisting of a sequence which anneals to a target mRNA and asequence for binding to dsDNA to form a triple helix.
 20. An isolatedsingle-stranded slDNA construct, which construct comprises a stem ofduplexed single-stranded DNA of annealed complementary bases of aninverted repeat joined at one end by a loop of non-duplexedsingle-stranded DNA, and having at the other end a single 5' terminusand single 3' terminus, and having a DNA antigene sequence positioned inthe non-duplexed portion of the slDNA; wherein the antigene sequence isselected from the group consisting of an antisense sequence for reducinggene expression and a sequence for binding to dsDNA to form a triplehelix.
 21. The slDNA of claim 20 wherein the antigene is a fragment forbinding to double-stranded DNA to form a triple helix.
 22. The slDNA ofclaim 21 wherein the sequence for binding to dsDNA to form a triplehelix is positioned in the loop of the slDNA.
 23. The slDNA of claim 20wherein the loop contains 4 to 20 bases.
 24. The slDNA of claim 20wherein both the 5' and 3' termini are free.
 25. The slDNA of claim 20wherein the loop contains 10 to 20 bases.
 26. The slDNA of claim whereinthe loop has 10 or more bases.
 27. The slDNA of claim 20 wherein theinverted repeat contains 6 to 20 bases.
 28. The slDNA of claim 20wherein the inverted repeat contains 10 to 30 bases.
 29. The slDNA ofclaim 20 wherein the inverted repeat contains 10 to 300 bases.
 30. TheslDNA of claim 20 wherein the antigene is positioned in the loop of theslDNA.
 31. The slDNA of claim 20 wherein the 5' terminus has asingle-stranded overhang with respect to the 3' terminus, or wherein the3' terminus has a single-stranded overhang with respect to 5' terminus;and the antigene is positioned in the single-stranded overhang of eitherthe 5' terminus or the 3' terminus.
 32. The slDNA of claim 20 whereinthe antigene is an antisense fragment for annealing to a target mRNA.33. The slDNA of claim 32 wherein the antisense fragment is positionedin the loop of the slDNA.